A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Chimpanzees, two in each group, were immunized with HCV-LP or HCV-LP adjuvant ASO1B. After four immunizations over an eight-month period, all animals developed strong HCV-specific cellular immune response including IFN-gamma and IL-2, CD4 and CD8 T-cell and proliferative lymphocyte responses against core, E1 and E2. The chimpanzees in both groups were challenged with a 100 CID50 of HCV CG1B inoculum. Upon challenge with HCV, one chimpanzee developed transient viremia with low HCV RNA titers (10E3-4 copies/ml) in the third and fourth weeks post-challenge. The three other chimpanzees became infected with higher levels of viremia (10E4-5 copies/ml) but their viral levels became unquantifiable (<1000 copies/ml) 10 weeks post-challenge. After HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral IFN-gamma T-cell and proliferative responses as well as the presence of intrahepatic T-cell response against the HCV structural proteins. These T-cell responses coincided with the fall in HCV RNA level. In comparison, four nave chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with viremia in the range of 10E5-6 copies/ml. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. The purity of HCV-LP after our current purification procedure is only about 10%, which needs to be significantly improved if this approach is going to be developed clinically as a vaccine candidate. Based on what we know about the structural features of HCV envelope proteins in the viral particles, we engineered a histidineX6 tag at the N-terminus of E2 protein, which allows surface exposure of this tag. The histidine sequences should not interfere with the structural assembly of HCV and would facilitate affinity purification. Expression of this tagged E2 in the context of HCV-LP demonstrated feasibility of this approach and further work is being performed to improve HCV-LP purification. In an effort to improve and broaden the immunogenicity of HCV-LP, we report the generation of novel chimeric hepatitis C virus-like particles carrying HCV nonstructural protein sequences with T-cell epitopes important for induction of protective immunity. Six highly-conserved HCV CD8+ T-cell nonstructural epitopes associated with viral clearance in humans were synthesized as a polytope construct and fused to the C-terminus of the E2 protein of HCV-LP. Hydrophobicity of the signal peptide sequence in the C-terminus of E2 was reduced and the polytope sequence was confirmed. Chimeric HCV-LP carrying the HCV nonstructural polytope were amplified in insect cells, purified, and characterized biochemically, immunologically and by electron microscopy. The immunogenicity of the chimeric HCV-LP was tested in AAD transgenic mice expressing the human HLA-A2.1 molecule. Similar to HCV-LP, chimeric HCV-LP induced HCV-specific humoral and cellular immune responses against the core and envelope. In addition, chimeric HCV-LP elicited robust T-cell responses against the nonstructural epitopes. The chimeric HCV-LP polytope strategy substantially improved and broadened the immunogenicity of HCV-LP and holds promise as a vaccine candidate against HCV infection in humans.In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen.